Immunology has recently provided vast improvements in clinical analysis because of refined techniques for detecting immunological complexes. Additionally, electrophoresis has provided enhanced methods for separating molecules having similar structures.
The immunoglobulins are a heterogeneous mixture of glycoproteins the basic monomeric units of which consist of two identical heavy (H) polypeptide chains and two identical light (L) polypeptide chains joined together by interchain disulfide bonds and noncovalent forces. There are five major classes of antibodies, IgM, IgD, IgG, IgE, and IgA. The differences among these antibodies derive from the different heavy (H) chains which comprise these antibodies.
Using IgG molecules of the same IgG subclass and allotype as an example, approximately one-half of the light chains and three-fourths of the heavy chains have amino acid sequences that are identical from one IgG molecule to the next. These regions of identical amino acid sequence are referred to as the "constant regions". The remaining one-half of the light chain and one-fourth of the heavy chain is composed of highly variable amino acid sequences and is referred to as the "variable region". These regions have also been found in the other immunoglobulin classes, IgA, IgM, IgD, IgE, and are the basis for immunoglobulin classification into three structural divisions:
(1) The constant region of the heavy chains determines the class distinction. These regions have been designated gamma (.gamma.), alpha (.alpha.), mu (.mu.), delta (.delta.), and epsilon (.epsilon.), determining IgG, IgA, IgM, IgD, and IgE, respectively.
(2) The light chain constant regions specify type and are designated kappa (.kappa.) or lambda (.lambda.).
(3) The variable or "idiotypic" regions of the heavy and light chains encompass the combining site or "complementarity-determining regions".
The highly variable regions of the heavy and light chains give rise to the antigen binding sites. Thus, changing the amino acid sequence in these variable regions yields antibodies with different antigenic specificities. The constant regions of the heavy chains are associated with activities such as complement fixation, cell membrane receptor interaction, passive cutaneous anaphylaxis, and transplacental transfer.
The immunoglobulins perform two functions within the immune response: recognition of antigens and initiation of a variety of secondary phenomena such as complement fixation and histamine release by mast cells. Four laboratory tests are commonly used to detect and quantify the immunoglobulins: serum protein electrophoresis (SPE), quantitation by immunodiffusion and nephelometry, immunoelectrophoresis (IEP), and immunofixation electrophoresis (IFE).
The initial step in the study of immunoglobinopathies is the SPE test. This screening procedure determines if the beta/gamma region of the pattern is abnormal in concentration or composition. A broad increase in the gamma-globulins on the electrophoretic pattern is indicative of numerous clones of plasma cells producing a heterogeneous mixture of immunoglobulins known as a polyclonal gammopathy. A polyclonal gammopathy is exhibited in a variety of clinical disorders, including autoimmune diseases, cancers, emphysema, and infections, to name a few.
Immunoelectrophoresis combines electrophoretic separation diffusion and immune precipitation of proteins. Both identification and approximate quantitation can thereby be accomplished for individual proteins present in serum, urine, or other fluid.
In the basic technique, a glass slide is covered with molten agar or agarose in a buffer solution. An antigen well and antibody trough are cut with a template cutting device. The serum sample (antigen) is placed in the antigen well and is separated in an electrical field with a potential difference of approximately 3.3 V/cm for 30-60 minutes. Antiserum is then placed into the trough, and both serum and antibodies are allowed to diffuse for up to 72 hours. The resulting precipitation lines may then be photographed or the slide washed, dried, and stained for a permanent record.
The two primary methods for classifying and typing monoclonal proteins are immunofixation and immunoelectrophoresis. In both of these methods an electrophoretic phase to separate the antigens of interest precedes the immunoprecipitation phase. Immunoelectrophoresis differs from immunofixation electrophoresis in the immunoprecipitation of the separated proteins. Immunoelectrophoresis places antibodies in a trough along the axis of electrophoretic migration. The separated antibodies and antigens are then allowed to diffuse through the support media. Where the diffusing antigen encounters the diffusing antibody, a precipitin arc is formed.
The interpretation of immunoelectrophoresis patterns depends on the comparison of arcs produced by a specimen with those produced by a normal control in the same IEP gel. Any deviations from these "normal" arcs are noted. These deviations are usually the absence or diminution of a precipitin arc, distortion of the arc in the form of thickening or bulging, or displacement of the arc from its normal position.
There are a number of immunoanalytical techniques in which a gel immunoprecipitation phase is preceded by zonal electrophoresis.
In zonal immunoprecipitation, first described by Poulik in 1952, (Poulik, M.D., Can. J. Med. Sci., 30:417-417 (1952)), zonally delineated immunoprecipitation is readily detectable in a blank layer of agar which is sandwiched between matching filterpaper strips respectively containing electrophoresed antigen (Diphtheria toxoid) and flocculating antiserum. This early study, although innovative, has been largely overlooked.
Transfer immunoelectrophoresis, described by Kohn in 1957, (Kohn, J., Nature, 180:986-987 (1957)), involved transferring a cellulose acetate zonal electrophoresis track to the surface of an agar plate, closely opposed with a parallel strip of precipitating antiserum. Formation of immunoprecipitation patterns is limited by the small amount of antiserum that can be applied via a surface strip, but can be strikingly enhanced by alternatively applying more antiserum via a conventional parallel trench.
Pizzolato et al, J. Immunol. Methods, 26:365-368 (1979), describe a cost-effective cellulose acetate microimmunofixation procedure.
Alper and Johnson, Vox Sang., 17, 445-452 (1969), described immunofixation electrophoresis based on the same principle as Poulik's zonal immunoprecipitation, but with the antiserum applied directly to the surface of an agarose-gel electrophoresis track. Optimal predilutions of antigen must be experimentally determined for each antiserum used.
Chuba, J. Appl. Biochem., 1, 37-50 (1979), proposed a double-diffusion-gradient immunoelectrophoresis using the same principles as the conventional immunoelectrophoresis of Graber and Williams, Biochem. Biophys. Acta, 10, 193-194 (1953) and Scheidegger, Int. Arch. Allergy Appl. Immunol., 7, 103-110 (1955), but with parallel antiserum troughs much closer to, and forming a 90.degree. angle with linear, rather than curved, fronts of antigen. In this technique, prozoning is minimized by the double diffusion gradient thus established. The prozone-minimizing effect of double immunodiffusion gradients was described by Elek, Brit. J. Exp. Pathol., 2:484-500 (1949) but has largely been overlooked.
Chuba (unpublished) has also proposed a transfer immunofixation electrophoresis based upon the same principle as Alper and Johnson, supra, but wherein the zonal agarose-gel track is transferred to matching rectangular wells containing precipitating antiserum. This permits immediate staining of the remaining tracks for reference zonal protein patterns.
In immunofixation electrophoresis (IFE), the degree of band detectability is highly dependent on the appropriateness of sample predilutions relative to the potency of each antiserum. In the case of double-diffusion gradient immunoelectrophoresis (DDG-IEP), conventional immunoprecipitation lines are readily detectable with most standard grade antisera over a broad range of very low to very high concentrations of antigen.
In IFE, prozoning due to excess of antigen or antibody is maximized because of direct confrontation of antigen versus antibody, and formation of crossed or spurred immunoprecipitation lines, as seen in double immunodiffusion systems, is abrogated by the in situ immunoprecipitation. In DDG-IEP and, to a lesser extent, in conventional IEP, however, prozoning is minimized, and crossed or spurred immunoprecipitation arcs classically associated with immunologic differences between mixed components are readily ascertainable. In one-dimensional double electroimmunodiffusion, also known as countercurrent immunoelectrophoresis, counterimmunoelectrophoresis, or electroprecipitation, the basic principle involves electrophoresis in a gel medium of antigen and antibody in opposite directions simultaneously from separate wells, with resultant precipitation at a point intermediate between their origins. The principal disadvantages of double diffusion without electromotive force are the time required for precipitation, about 24 hours, and the relative lack of sensitivity. Double electroimmunodiffusion in one dimension can produce visible precipitin lines within 30 minutes, and is approximately ten times more sensitive than standard double diffusion techniques. However, this technique is only semiquantitative.
One-dimensional single electroimmunodiffusion is also known as rocket electrophoresis, or the Laurell technique. The principal application of this technique has been to quantitate antigens with faster electrophoretic mobility than immunoglobulins. In this technique, antiserum to the particular antigen or antigens one wishes to quantitate is incorporated into an agarose supporting medium on a glass slide in a fixed position so that antibody does not migrate. The specimen containing an unknown quantity of the antigen is placed into a small well. Electrophoresis of the antigen into the antibody-containing agarose is then performed. The resultant pattern of immunoprecipitation resembles a spike or rocket, which led to the term "rocket electrophoresis".
The rocket pattern occurs because precipitation occurs along the lateral margins of the moving boundary of antigen as the antigen is driven into the agar containing the antibody. Gradually, as antigen is lost through precipitation, its concentration at the leading edge diminishes, and the lateral margins converge to form a sharp point. The total distance of antigen migration for a given antiserum concentration is linearly proportionate to the antigen concentration. The sensitivity of this technique is approximately 50. micrograms/ml for proteins. Unfortunately, the weak negative charge of immunoglobulins prevents their electrophoretic mobility in this system unless special electrolytes and agar are used. Recently, several commercially available systems have been introduced for quantitating serum immunoglobulins and complement components with this technique.
Immunoelectrophoresis can aid in distinguishing polyclonal from monoclonal increases in gamma-globulin. Additionally, decreased or absent immunoglobulins observed in various immune deficiency disorders can be analyzed with this technique. However, a further quantitative analysis such as single radial diffusion, nephelometry, or radioimmunoassay should be performed for measurement of immunoglobulin levels other than in the case of electrophoretically well-delineated monoclonal bands, which can be quantitated by scanning densitometry.
Immunoelectrophoresis is also of great practical benefit in identifying abnormal immunoglobulin components, such as monoclonal Bence-Jones proteins, in the urine of patients with plasma cell dyscrasias or certain autoimmune disorders. Thus, with specific anti-kappa and anti-lambda antisera, the monoclonality of Bence-Jones protein in urine can be readily ascertained.
Antisera to "free light chains" (kappa or lambda) obtained from the urine of myeloma patients may occasionally reveal antigenic determinants not detectable on light chains "bound" to heavy chains. In H chain diseases, fragments of the immunoglobulin H chain are present in increased amounts in the serum and urine. Immunoelectrophoresis is also helpful in identifying abnormal immunoglobulin patterns (e.g., oligoclonal gamma banding) in the cerebrospinal fluid of patients with various neurologic diseases.
Immunofixation electrophoresis involves zonal separation of proteins electrophoretically in an appropriate support matrix, followed by immunoprecipitation in situ with monospecific antisera. Nonprecipitated proteins are removed by washing and the immunoprecipitation bands are revealed with a protein stain. This method has been used clinically to identify C3 conversion products and to identify paraproteins. The latter is especially helpful for low-level IgM or IgA components, which may be buried in an excess of normal IgG. There are several modifications of this basic method, such as overlay with radioactive or enzyme-linked antibodies that markedly increase the sensitivity of the method.
It can thus be seen that chemical laboratory procedures for evaluating serum and other fluids for possible presence of paraproteins are very important but are not widely enough utilized due to the difficulties and drawbacks of the present procedures.
Ideally, all immunoprecipitation patterns obtained following an initial electrophoretic separation phase should be evaluated within the context of appropriate immunoprecipitation control patterns, as well as well-delineated zonal protein reference patterns run in the same plate. In this regard, conventional immunoelectrophoresis includes normal immunoprecipitation patterns as controls in each plate, but does not provide zonal protein reference patterns. The latter, even if included in each run, would tend to be poorly delineated due to the traditional manner in which immunoelectrophoresis samples are applied via circular openings. Immunofixation electrophoresis, on the other hand, provides well-delineated zonal protein reference patterns in each plate but does not readily lend itself to the inclusion of normal immunoprecipitation control patterns.
It would be highly advantageous to have a single-plate procedure for pairing immunoelectrophoretic patterns with matching zonal protein patterns in order to provide both immunoprecipitation control patterns and well-delineated zonal protein reference patterns on the same plate.